Methods and apparatus for measuring analytes using large scale FET arrays

Abstract

Methods and apparatus relating to very large scale FET arrays for analyte measurements. ChemFET (e.g., ISFET) arrays may be fabricated using conventional CMOS processing techniques based on improved FET pixel and array designs that increase measurement sensitivity and accuracy, and at the same time facilitate significantly small pixel sizes and dense arrays. Improved array control techniques provide for rapid data acquisition from large and dense arrays. Such arrays may be employed to detect a presence and/or concentration changes of various analyte types in a wide variety of chemical and/or biological processes. In one example, chemFET arrays facilitate DNA sequencing techniques based on monitoring changes in hydrogen ion concentration (pH), changes in other analyte concentration, and/or binding events associated with chemical processes relating to DNA synthesis.

1. A method for nucleic acid sequencing, comprising:
 disposing a plurality of template nucleic acids in at least 10 5  reaction volumes for at least 10 5  sensors, the sensors comprising field-effect transistors having floating gates coupled to the reaction volumes to detect hydrogen ions liberated through incorporation of a nucleotide into one or more primers that are hybridized to at least one of the plurality of template nucleic acids; 
 introducing a known nucleotide into the reaction volumes; 
 coupling circuitry to field-effect transistors of the sensors to measure pH changes within the reaction volumes within a pre-specified pH range that is less than a maximum pH range detectable by the field-effect transistors, the measured pH change based on liberation of hydrogen ions detected by the field-effect transistors resulting from incorporation of the introduced known nucleotide into the one or more primers, and the circuitry further to generate output signals indicating the measured pH changes; and 
 analyzing the output signals generated by the circuitry to determine at least a portion of sequences corresponding to a portion of the template nucleic acids. 
 
     
     
       2. The method of  claim 1 , wherein the sensors respectively occupy an area of 100 μm 2  or less and have a pitch of 10 μm or less and wherein the reaction volumes have respective volumes in the range of from 1 μm 3  to 1500 μm 3 . 
     
     
       3. The method of  claim 1 , wherein the reaction volumes respectively contain at least 10 5  copies of one of said template nucleic acids. 
     
     
       4. The method of  claim 1 , wherein the nucleic acid templates are retained by at least one solid support. 
     
     
       5. The method of  claim 1 , wherein the reaction volumes and the sensors are each greater than 256,000. 
     
     
       6. The method of  claim 4 , wherein the at least one solid support comprises at least one particle. 
     
     
       7. The method of  claim 6 , wherein the at least one particle comprises cellulose, a cellulose derivative, gelatin, an acrylic resin, glass, silica gel, polyvinyl pyrrolidine (PVP), co-polymers of vinyl and acrylamide, polystyrene, polystyrene cross-linked with divinylbenzene, a polyacrylamides, a latex gel, dextran, a crosslinked dextrans, rubber, silicon, a plastic, nitrocellulose, a natural sponge, a metal, agarose gel, or a streptavidin coating. 
     
     
       8. The method of  claim 6 , wherein the at least one particle is 1-10 μm in diameter. 
     
     
       9. The method of  claim 1 , wherein the known nucleotide is a nucleotide selected from the group consisting of dATP, dCTP, dGTP and dTTP. 
     
     
       10. The method of  claim 1 , wherein introducing the known nucleotide comprises delivery of a wash buffer solution comprising ATP to the reaction volumes. 
     
     
       11. The method of  claim 1 , further comprising:
 washing unincorporated introduced known nucleotide from the reaction volumes; 
 introducing a known second nucleotide into the reaction volumes; 
 coupling the circuitry to the field effect transistors to measure second pH changes within the reaction volume based on liberation of hydrogen ions detected by the field-effect transistors resulting from incorporation of the introduced known second nucleotide into the one or more primers, and the circuitry further generating second output signals indicating the measured second pH changes; and 
 analyzing the second output signals to determine at least a second portion of sequences corresponding to a second portion of the template nucleic acids. 
 
     
     
       12. The method of  claim 1 , wherein the measured pH changes are based on threshold voltages of the field-effect transistors due to the liberated hydrogen ions. 
     
     
       13. The method of  claim 1 , wherein coupling the circuitry to a given field-effect transistor of a given sensor in the sensors comprises:
 coupling a first terminal and a second terminal of the given field-effect transistor to bias circuitry, the bias circuitry applying a read voltage to the first terminal and establishing a voltage difference between the first terminal and the second terminal; 
 coupling the given field-effect transistor to a current source to provide a bias current through the given field-effect transistor, thereby establishing a voltage level at the second terminal based on a threshold voltage of the given field-effect transistor resulting from a corresponding pH change; and 
 coupling the second terminal to readout circuitry to read the voltage level at the second terminal to generate a corresponding output signal. 
 
     
     
       14. The method of  claim 13 , wherein the given sensor further comprises:
 a first switch coupled between the second terminal of the given field-effect transistor and the bias circuitry and the readout circuitry; and 
 a second switch coupled between the second terminal of the given field-effect transistor and the current source. 
 
     
     
       15. The method of  claim 14 , wherein the first switch and the second switch are responsive to a common select signal to couple the second terminal of the given field-effect transistor to the bias circuitry and to the current source. 
     
     
       16. The method of  claim 14 , wherein the first switch and the second switch are each single-transistor switches having channel types the same as that of the given field-effect transistor. 
     
     
       17. The method of  claim 14 , wherein the first terminal of the given field-effect transistor is directly connected to the bias circuitry. 
     
     
       18. The method of  claim 14 , wherein the bias circuitry and the readout circuitry is shared among a plurality of the sensors. 
     
     
       19. The method of  claim 18 , wherein the sensors are arranged in a plurality of rows and a plurality of columns, and the bias circuitry and the readout circuitry is shared among a column of sensors in the plurality of columns. 
     
     
       20. The method of  claim 1 , wherein a given sensor of the sensors further comprises:
 a first single-transistor switch coupled between a given field-effect transistor of the given sensor and a current source to provide a bias current through the given field-effect transistor to establish a voltage level on a first terminal of the given field-effect transistor based on the pH change; and 
 a second single-transistor switch coupled between the first terminal of the given field-effect transistor and the circuitry to read the voltage level at the first terminal to generate a given output signal. 
 
     
     
       21. The method of  claim 20 , wherein the first single-transistor switch and the second single-transistor switch have channel types the same as that of the given field-effect transistor. 
     
     
       22. The method of  claim 20 , wherein body connections of the first single-transistor switch, the second single-transistor switch and the given field-effect transistor are each coupled to a common line. 
     
     
       23. The method of  claim 1 , wherein the pre-specified pH range extends between pH 7 and pH 9. 
     
     
       24. The method of  claim 1 , wherein the pre-specified pH range corresponds to 2 pH units. 
     
     
       25. The method of  claim 1 , wherein the floating gates of the field-effect transistors are coupled to the reaction volumes via a passivation layer sensitive to hydrogen ions. 
     
     
       26. The method of  claim 1 , wherein the disposing includes disposing a template nucleic acid into a reaction volume, and amplifying the template nucleic acid in the reaction volume. 
     
     
       27. The method of  claim 26 , wherein the amplifying includes isothermally amplifying the template nucleic acid in the reaction volume.